Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China

Bacillus thuringiensis isolates from different ecological regions and sources of China were analyzed to study the distribution and diversity of cry genes and to detect the presence of novel cry genes. Strains containing cry1-type genes were the most abundant and represent 237 of the 310 B. thuringiensis isolates (76.5%). About 70 and 15.5% of the isolates contained a cry2 gene or cry9 gene, respectively, while 10.0% of the strains did not contain a cry1, cry2, or cry9 gene. Among the cry1 containing isolates, cry1A (67.7%), cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%) genes were the most abundant. Forty-three different cry1 gene profiles were detected in this collection. Several cry1 genes were associated at a high frequency, such as the cry1C-cry1D and cry1A-cry1I gene combination. The cry1A and cry2 amplicons were digested with selected restriction enzymes to examine sequence diversity. Based on this RFLP analysis, one novel cry1A-type gene was observed.     

Cloning of cry2Aa and cry2Ab genes from new isolates of Bacillus thuringiensis and their expression in recombinant Bacillus thuringiensis and Escherichia coli strains

Bacillus thuringiensis (Bt) is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of Bt are promising candidates for management of resistance development in insects due to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. Two insecticidal crystal protein genes of Bt, viz. cry2Aa and cry2Ab were cloned from new isolates of Bt, 22-4 and 22-11, respectively. Expression of both the genes was studied in an acrystalliferous strain of Bt (4Q7) by fusing the cry2Aa and cry2Ab genes downstream of cry2Aa promoter and orf1 + orf2 sequences. Western blot analysis revealed a low level expression of the cloned cry2Aa and cry2Ab genes in the recombinant Bt strains. High-level expression of cry2Aa and cry2Ab genes was achieved in the recombinant E. coli by cloning the cry2A genes under the control of the T7 promoter.     

Screening and characterization of Bacillus thuringiensis isolates from Brazil for the presence of coleoptera-specific cry genes

Forty-three Bacillus thuringiensis isolates from Brazil and 3 from Argentina were screened, using the polymerase chain reaction (PCR), for various coleoptera-specific cry genes. Seven isolates produced specific and/or nonspecific DNA fragments in a PCR reaction with primers specific for two coleopteran cry genes and 4 of these produced DNA fragments with primers specific for 7 known coleopteran cry genes. These isolates showed, by electron microscopy, the presence of spherical crystals. They also showed proteins of around 70 kDa which were immunologically similar to the Cry3Aa protein from B. thuringiensis subsp. tenebrionis. The 3 isolates from Argentina were toxic to T. molitor, and although no isolate from Brazil showed toxicity, they might show toxicity to another insect species.     

Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis delta-endotoxin nomenclature committee.     

PCR-based method for the detection of cry genes in local isolates of Bacillus thuringiensis from Thailand

A total of 134 isolates of Bacillus thuringiensis obtained from different geographical and ecological origins in Thailand were analyzed to determine the distribution and diversity of cry1, cry2 and cry9 genes encoding for Cry proteins toxic to lepidopteran insects. Strains containing cry1-type genes (109/134 or 81.3%) were found at the same frequency as strains harboring cry2 gene (108/134 or 80.6%) whereas only 50 strains contained cry9 gene (50/134 or 37.3%). Seventeen percent (23/134) of the B. thuringiensis isolates did not harbor any cry1, cry2 or cry9 genes. Among cry1 containing isolates, cry1A (49.3%), cry1B (50.0%), cry1G (48.5%), cry1I (49.3%), cry1J (35.1%) and cry1L (47.0%) were considered abundant. The cry2 gene was distributed with high frequency (>70%) in every region of the country. The study of cry gene combinations revealed 14 cry gene profiles.     

Characterization and cloning of the cry2A gene from indigenous isolates of Bacillus thuringiensis

Molecular Biology - Discovery of novel cry genes of Bacillus thuringiensis (Bt) with higher toxicity is important for the development of transgenic Bt crops resistant to target pests. Two new...     

Diversity analysis and characterization of Coleoptera-, Hemiptera- and Nematode-active cry genes in native isolates of Bacillus thuringiensis

Applications to combat non-lepidopteran insects are not as common as applications against lepidopteran insects. The aim of the present work was to isolate and identify Bacillus thuringiensis isolates from soil samples using five approaches, viz., analysis of crystal protein production by microscopy; detection of cry gene content by PCR, SDS-PAGE profiling; cloning and sequencing; phylogenetic analysis; and toxicity testing. Two hundred soil samples were used for isolation of B. thuringiensis and a total of 69 putative isolates of B. thuringiensis that produce parasporal crystalline inclusions were isolated from 5,267 Bacillus-like colonies. A bipyramidal inclusion was predominant in 32.2 % of the B. thuringiensis isolates compared to other shapes. Crystal protein profiling of B. thuringiensis isolates by SDS-PAGE analysis showed the presence of bands of 130, 73, 34, 25 and 13 kDa, among which 50–60 kDa bands were present abundantly. PCR analysis revealed the predominance of Coleopteran-active cry genes in these isolates. Variation in nucleotide sequences, crystal morphology and mass of crystal protein(s) purified from the isolates of B. thuringiensis revealed genetic and molecular diversity. Four strains containing Coleopteran-active cry genes showed higher toxicity against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) adults when compared with B. thuringiensis subsp. morrisoni pathovar tenebrionis. These results are useful in emphasizing the distribution of cry genes and for prognostication of toxicity, and may contribute to the identification of novel candidate genes for bioengineered crop protection.     

苏云金芽孢杆菌cry基因研究进展

从cry基因的分类、cry基因与转座因子的关系、cry基因的表达调控以及cry基因的作用机理等 4个方面综述了cry基因的研究进展 ,并简要展望了其研究和应用前景。     

Characterization of cry9Da4, cry9Eb2, and cry9Ee1 genes from Bacillus thuringiensis strain T03B001

Three cry9 genes, cry9Da4, cry9Eb2, and cry9Ee1, were cloned from Bacillus thuringiensis strain T03B001 using a high-resolution melting analysis method. All three cry9 genes were overexpressed in Escherichia coli Rosetta (DE3), and the expressed products Cry9Eb2 and Cry9Ee1 were shown to be toxic to Plutella xylostella and Ostrinia furnacalis, but not to Helicoverpa armigera or Colaphellus bowringi. The bioassay of Cry9Eb2 and Cry9Ee1 against Cry1Ac-resistant P. xylostella strains indicated that both novel Cry9 toxins exhibited no cross-resistance with Cry1Ac. Cry9Eb2 and Cry9Ee1 can be applied not only for P. xylostella and O. furnacalis control, but also for the Cry1Ac-resistance management of pests.     

Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cry1 genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.     

Expression of orf1 from the Bacillus thuringiensis NRD-12 cry2Aa1 Operon

The 5′ untranslated region and the orf1 sequence from the cry2Aa1 operon from Bacillus thuringiensis subsp. kurstaki NRD-12 were sequenced and compared to that from strain HD-1. The start codon described in HD-1 does not yield in NRD-12 a protein of the expected size of 20 kDa, but a 10-amino acid peptide. A second, highly conserved start codon is located 25 bp downstream from the first one and corresponds to an open reading frame of the same size in all known orf1-related sequences. Expression of lacZ gene fusions created at the level of the first ATG, second ATG, and stop codon of the NRD-12 orf1 sequence showed that orf1 is translated from the second ATG. The expected protein is 19 kDa in size. The expression starts at t2, which is in agreement with the presence of a BtI promoter in the cry2Aa1 operon. Received: 8 January 1999 / Accepted: 9 February 1999     

Agrobacterium-mediated transformation of Brassica campestris ssp. Parachinensis with synthetic Bacillus thuringiensis cry1Ab and cry1Ac genes

 An effective plant regeneration procedure and a gene transfer system via Agrobacterium tumefaciens were developed in Brassica campestris ssp. parachinensis. Hypocotyls from 5-day-old seedlings with 2 days pre-culture were infected with Agrobacterium strain MOG301 harboring a binary vector containing a synthetic Bacillus thuringiensis (B.t.) cry1Ab or cry1Ac gene with full codon-modification. After culture and selection on MS medium supplemented with 4.0 mg/l BAP, 2.0 mg/l NAA, 70 μM AgNO₃ and 50 mg/l kanamycin, a number of kanamycin-resistant plantlets were regenerated. PCR and Southern blotting analysis were used to identify and characterize the transgenic plants with the integrated cry1Ab or cry1Ac gene. Western blotting analysis of the transgenic plants confirmed the expression of insecticidal proteins encoded by cry1Ab or cry1Ac. Subsequent bioassay with larvae of the Diamondback moth, Plutella xylostella, demonstrated that the transgenic plants were resistant to feeding damage. Received: 22 February 1999 / Revision received: 14 April 1999 / Accepted: 26 April 1999     

Identification and molecular characterization of novel cry1-type toxin genes from Bacillus thuringiensis K1 isolated in Korea

To clone novel cry1-type genes from the Bacillus thuringiensis K1 isolate, about 2.4-kb-long PCR fragments were amplified with two primer sets of ATG1-F/N400-R and 1BeATG1-F/N400-R. Using PCR-RFLP, three novel cry1-type genes, cry1-1, cry1-7, and cry1-44, were obtained from B. thuringiensis K1 and the complete coding sequences of these novel genes were analyzed. The Cry1-1, Cry1-7, and Cry1-44 proteins showed maximum similarities of about 78.0%, 99.7%, and 91.0% with the Cry1Ha1, Cry1Be1, and Cry1Ac2 proteins, respectively. These novel cry1-type genes were expressed using a baculovirus expression vector system and their insecticidal activities were investigated. Whereas all three novel genes were toxic to Plutella xylostella larvae, only Cry1-1 showed insecticidal activity against Spodoptera exigua larvae.     

PCR-based approach for detection of novel Bacillus thuringiensis cry genes.

A two-step strategy, named exclusive PCR or E-PCR, has been developed to overcome the main limitation of PCR, which is the detection of already-known sequences only. This strategy allows the ability to detect and further clone and sequence genes for which no specific primers are available and in which a variable region exists between two conserved regions. This approach has been applied to Bacillus thuringiensis cryI genes by the use of mixtures of degenerate and specific primers recognizing well-known sequences. The first step allows the accurate identification of already-characterized cryI genes by the use of three primers. During the second step, the same sets of primers are used to exclude known sequences and to positively detect cryI genes unrecognized by any specific primer. The method, as well as its application to detect, clone, and sequence a novel cryIB gene, is described in this article.     

苏云金芽胞杆菌cry2Ab、cry9Ea基因的表达及活性分析

旨在为农业害虫防治提供更多的苏云金芽胞杆菌基因资源,从中国吉林市龙潭山土壤样品中分离得到野生菌株命名为Bt LTS-7,扫描电镜显示该菌株产生晶体形状为双锥体和正方体,聚丙烯酰胺凝胶电泳显示该菌株产生130 kD和71 kD的晶体蛋白,通过PCR鉴定出该菌株中含有cry2Ab和cry9Ea基因,并成功克隆到了这两个新基因,并被Bt国际命名委员会正式命名为cry2Ab28和cry9Ea9,将两个基因分别在大肠杆菌Rosetta( DE3)中表达,并进一步对其表达蛋白进行杀虫活性测定.结果显示,Cry2Ab28蛋白对棉铃虫(Helicoverpa armigera)初孵幼虫具有杀虫活性,LC50为32.45 μg/mL.Cry9Ea9蛋白对小菜蛾初孵幼虫(Plutella xylostella)具有较高杀虫活性,LC50为0.77μg/mL.     

Identification of cry1I-Type Genes from Bacillus thuringiensis Strains and Characterization of a Novel cry1I-Type Gene

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis δ-endotoxin nomenclature committee.     

Contents of cry genes and insecticidal toxicity of Bacillus thuringiensis strains from terrestrial and aquatic habitats

AIMS: Two Bacillus thuringiensis collections from terrestrial and aquatic habitats were compared in order to study the possible interrelationships between habitat and biological characteristics (serovar, cry genes content and toxicity). METHODS AND RESULTS: Bacillus thuringiensis strains were characterized by serology, PCR, and one-dose treatment against the noctuids Helicoverpa armigera and Spodoptera exigua, and the dipteran Tipula oleracea. A total of 12 and 10 different serovars were identified within terrestrial and aquatic strains, respectively. The number of non-toxic strains was greater in aquatic (41.6%) than in terrestrial habitats (5.3%). The genes cry1C, cry1D and cry1E were significantly more frequent in the terrestrial habitat. The cry1B gene was very frequent within thuringiensis strains. CONCLUSIONS: A high diversity was found in terms of serovars present and cry genes content in both collections. The relative frequency of individual cry genes was different in both collections, and a serovar-dependent distribution of the cry1B gene was found. Some strains sharing the same set of cry genes differed in their toxicity, suggesting important differences in gene expression. SIGNIFICANCE AND IMPACT OF THE STUDY: The inter-relationships between serology, cry gene content and toxicity may allow a better understanding of B. thuringiensis ecology.     

Molecular typing of native Bacillus thuringiensis isolates from diverse habitats in India using REP-PCR and ERIC-PCR analysis

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. The subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. Therefore molecular typing and diversity analysis of B. thuringiensis has enormous importance for discrimination of strains isolated from different sources. In this study, 113 native B. thuringiensis isolates collected from diverse habitats and locations in India and 27 B. thuringiensis type strains obtained from the Bacillus Genetic Stock Centre (BGSC), Ohio State University, USA and used as reference, were analyzed for molecular typing. Genotypic data of 140 B. thuringiensis isolates and type strains was generated by using REP-PCR and ERIC-PCR primers and unweighted pair group method with arithmetic mean (UPGMA) analysis using NTSYSpc2.2 and grouped into 4 main clusters. All the groups have isolates from diverse origins. No group was found to represent any specific origin or location. The observed patterns of REP-PCR and ERIC-PCR pattern were discriminatory enough to reveal differences in the B. thuringiensis isolates and reference strains. The resolution power and marker index of the ERIC-PCR (RP 9.39, MI 6.34) was found to be higher than that of the REP-PCR (RP 6.20, MI 4.48). The REP-PCR and ERIC-PCR markers have been found to be useful for discrimination of B. thuringiensis isolates and reference strains. ERIC-PCR was the more informative of the two techniques. This study showed that the B. thuringiensis isolates collected from diverse habitats in India had a high degree of genetic diversity.     

Composition and ecological distribution of cry proteins and their genotypes of Bacillus thuringiensis isolates from warehouses in China

The composition and distribution of insecticidal crystal proteins (Cry proteins) and their genotypes of Bacillus thuringiensis isolates from warehouses were evaluated through SDS-PAGE and PCR techniques. The results showed that the electrophoretic patterns of delta-endotoxin crystal preparations were divided into five types. The isolates containing approximately 135 kDa with a 65-kDa protein or only a approximately 135-kDa protein, which amounted to 55.74 and 35.25% of all isolates respectively, were the two major profiles of Cry protein isolated. The distribution of cry genes of B. thuringiensis from warehouses was highly variable. Cry protein genotypes detected in B. thuringiensis isolates included cry1Aa5, cry1Ab9, cry1Ac5, cry1Ba, cry1Ca1, cry1Da1, cry1Ea3, cry2, and cry3 genes, but not cry1Fa2. Among them, cry2, cry1Ac5, and cry1Ab9 genes were the most common in our B. thuringiensis isolates. Most B. thuringiensis isolates contained several cry genes in a total of 18 profiles. Among them, cry1Ac5 with cry1Ea3; cry1Aa5, cry1Ab9, cry1Ac5 with cry1Ea3; and cry1Aa5, cry1Ab9 with cry1Ac5 were the three principal profiles. The distribution of the Cry proteins and cry genes in isolates depended on geography and type of warehouses. Gene profiles may be used as markers for insecticidal activity of B. thuringiensis strains, but they did not directly reflect the toxic level of B. thuringiensis strains. The serotype of B. thuringiensis strains did not directly reflect the specific cry gene profiles in the strains, but certain relationships can be established between the serotype and cry genotype.